The long-term goal of this work is to understand how Arp2/3 complex and its regulators control the assembly and disassembly of actin networks. Arp2/3 complex is an essential component of the actin assembly machinery because of its ability to nucleate branched actin filaments in response to cellular signals. Arp2/3 complex is involved in a number of processes that occur in healthy cells, such as endocytosis and motility of growth cones, but also plays roles in host cell infection by bacterial pathogens and the metastasis of tumor cells. Therefore, advances in understanding cellular control of branched actin networks created by Arp2/3 complex will provide a foundation to advance treatments of bacterial infections and cancer. The activity of Arp2/3 complex is tightly regulated in vivo, and the vast majority of studies on Arp2/3 complex activation have focused on WASP family proteins. WASP proteins require preformed filaments to activate Arp2/3 complex and propagate branched networks, but few studies have addressed the source of preformed filaments to initiate branching. We recently discovered that WISH/DIP/SPIN90 (WDS) proteins are key branched actin network initiators that can seed WASP-mediated branching by activating Arp2/3 complex without preformed filaments. Despite the fact that initiation is perhaps the most important step in controlling branched network assembly, how WDS proteins activate the complex to create seed filaments is unknown. Furthermore, it is not understood how the activity of WDS proteins is coordinated with other activators like WASP, or how WDS proteins themselves are regulated. Here we propose to determine the molecular mechanisms by which WDS proteins activate Arp2/3 complex, alone and coordinately with WASP. In addition, we will elucidate how WDS proteins are regulated, which will allow us to understand the molecular basis for branched actin network initiation. Completion of these studies will provide a significant advance in our understanding of cellular control of the actin cytoskeleton. We will pursue these goals through the following three specific aims: 1.) Define structural features in WDS proteins required for Arp2/3 complex activation; 2.) Determine how WDS proteins interact with Arp2/3 complex and influence its conformation to stimulate activation; and 3.) Determine how WDS proteins are regulated. To accomplish these aims we will use a combination of x-ray crystallography, biochemical and biophysical assays, mathematical modeling, single molecule total internal reflection fluorescence microscopy and fluorescence imaging of live S. pombe cells.